The cytoplasmic granules purified from rat large granular lymphocyte tumors with NK activity have been studied to determine their role in cytotoxic and other functions of these lymphocytes. In addition to the major lytic protein termed cytolysin, we have found a series of protease activities present in the purified dense granules. Two of these, which show potent activity against the trypsin thioester substrate known as BLT, have been purified to homogeneity and are among the major granule proteins. One of these enzymes is a disulfide linked dimer of two roughly 30kd protein chains, while the other is comprised of a single chain of about 27kd. These enzymes do not readily hydrolyze protein substrates cleaved by trypsin, but can be shown to act on several synthetic peptide p-nitroanilide substrates with arginine at the P1 site. Inhibitor studies show that both enzymes are serine proteases with a pH optimum of about 8. The pattern of activity inhibition by a panel of inhibitors on the two enzymes is distinct, indicating that these two enzymes do not share a simple monomer-dimer relationship. In addition to these two BLT-hydrolyzing enzymes which have been purified, granule extracts separated by gel filtration show an additional peak of activity against tryptic p-nitroanilide substrates, and 2-3 peaks of activity against chymotryptic p-nitroanilide substrates. Although the physiological roles of these serine proteases are still unclear, each of the two purified BLT-hydrolysing enzymes can synergistically enhance the lysis of nucleated cells by the purified granule cytolysin. Studies of granule proteoglycans have been undertaken by labeling the growing LGL tumors in vivo with 35S-S04. It was found that this label is localized in both dense (cytolysin positive) and light (lytically inactive) granule fractions of Percoll fractionated homogenates; kinetic studies suggest that the light granule fraction is a precursor of the dense granule fraction. Western blots of Percoll gradients with rabbit anti-cytolysin antibody show that the cytolysin protein is present in the light granule fraction in spite of the lack of dectable activity.